Comparative phenotypic, functional, and molecular analysis of pluripotent stem cells arguably the most significant challenge in stem cell biology today is determining whether human induced pluripotent stem cells (hiPSC) are truly equivalent to human embryonic stem cells (hESC), and thus can serve as a stable, safe, and less controversial resource for basic research and cell replacement therapies. Although the evidence to date suggests that murine ESC and iPSC are functionally interchangeable, the analysis is preliminary and incomplete, especially for human cells, and evidence is accumulating that important molecular differences distinguish the two types of pluripotent stem cells. To answer whether significant functional and molecular differences exist, we have assembled an outstanding cohort of collaborators, each with unique and complementary expertise, to perform a comprehensive phenotypic, functional, and molecular comparison of multiple ESC and iPSC lines using genomic, epigenetic, proteomic, computational, and pathologic analysis. In recent years, the Daley lab has simultaneously pursued derivation of novel hESC from discarded IVF embryos and generation of hiPSC by direct somatic cell reprogramming. Moreover, we have an extensive collection of murine pluripotent stem cells generated by direct reprogramming (miPSC) or isolated from embryos after fertilization (fESC), parthenogenesis (pESC), and somatic cell nuclear transfer (ntESC). This comprehensive set of reagents affords us a unique opportunity to test the hypothesis that iPSC are the functional equivalents of ESC in assays of pluripotency, but that molecular differences persist between the two classes of pluripotent stem cells. We will further test the hypothesis that differences between ESC and iPSC are largely due to residual transgene expression in iPSC, and will resolve once transgenes are removed. However, preliminary data suggests that even transgene-free iPSC are epigenetically distinct from ESC. Thus, an alternative hypothesis is that factor-based reprogramming leaves a residual epigenetic signature of the tissue of origin ("epigenetic memory"), and that the reprogramming process confers unique molecular features on iPSC. In addition to comparing and contrasting ESC and iPSC, our analysis will illuminate the degree of variation among independent clones of ESC and iPSC. Defining the extent of functional similarity, assessing the nature of any molecular differences, and defining biomarkers of the successfully reprogrammed state are key goals of this proposal. Insights gleaned herein will contribute to improved reprogramming methods, thereby facilitating the application of iPSCs to disease research and cell therapies. PUBLIC HEALTH RELEVANCE: Pluripotent stem cells offer tremendous promise as tools for basic biomedical research, disease modeling and drug screening, and provide a means of deriving patient-specific rejection-proof cells that might be used in cell replacement therapies for a large number of genetic, malignant, and degenerative diseases. Techniques for establishing "induced pluripotent stem" or "iPS" cells fulfill the long-sought strategy for generating customized stem cells. If proven equivalent-both functionally and molecularly-to blastocyst-derived human embryonic stem cells, iPS cells will facilitate research, quell contentious public debate, and yield a new modality for tissue repair and regeneration.